This grant application focusses on cardiac macrophages, which are an integral stromal component of the myocardium. While precise functions of resident versus recruited heart macrophages are still being unraveled, epidemiological data document that numbers of monocytes, the blood progenitors of inflammatory infarct macrophages, correlate closely with cardiovascular mortality: the higher the number of monocytes, the worse the patient's prognosis. The growing understanding of the cell's disease-promoting functions suggest that tissue macrophage numbers predict outcome even more accurately; however, since we currently lack quantitative tools to detect macrophages in the heart, we cannot measure macrophages in diseased tissues, a limitation that impedes emerging immune-targeting therapeutics in cardiovascular diseases. Cardiac and vascular biopsies are invasive, prone to sampling error and often not feasible, but quantitative imaging could measure macrophage numbers body-wide and non-invasively, thereby detecting disease foci to guide therapy and enable immunomodulatory drug trials. We propose to address the unmet medical need of noninvasive macrophage quantification in ischemic heart disease by deploying clinically viable nanoparticles for macrophage imaging at cellular and organ levels. Based on extensive profiling studies, we have recently developed a lead formulation with unprecedented macrophage uptake and surprisingly fast pharmacokinetics. The 6 nm carboxymethyldextran-based polymeric nanoparticle (DNP) is available in 2 versions: i) fluorescent (DNP-VT680) for microscopic imaging, which enables us to study in vivo macrophage biology at cellular resolution, and ii) labeled with fluorine-18 (18F-DNP), for translational macrophage PET imaging. These two DNP versions allow us to ask basic biological questions at the single-cell level and then extrapolate findings to clinical scenarios. We developed facile DNP labeling with the widely-available isotope fluorine-18, a strategy inspired by our ultimate goal of using the material in humans with acute myocardial infarction (MI). We propose to advance this technology by i) testing DNP's efficiency for answering relevant questions on macrophage biology in mouse models of cardiovascular disease using intravital microscopy of DNP-VT680 and ii) confirming the material's value in translational large animal MI studies. In 3 Aims, we will validate the material for macrophage PET/MRI in mice with myocardial infarction, test the capabilities of DNP for monitoring innovative macrophage-targeted therapeutics and use 18F-DNP for PET/MRI of macrophages in swine with acute MI. These studies will chaperone 18F-DNP, our most promising PET nanoparticle, towards clinical translation, addressing the unmet clinical need to detect inflammatory macrophages in ischemic heart disease.